Normal human cells have a limited capacity to proliferate, a process termed replicative aging. Increasing evidence over the last decade has implicated telomeres, the structures that cap the ends of the chromosomes, as the molecular clock that counts the number of times the cell has divided. The mechanism of lagging strand DNA synthesis prevents DNA polymerase from replicating the DNA all the way to the 5' end of a linear chromosome, leaving a 3' overhang and causing the chromosomes to shorten every time a cell divides. Human telomeres are composed of many kilobases of the repetitive sequence TTAGGG that together with telomere binding proteins prevent the cell from recognizing the end of the chromosome as a DNA break needing repair. Cellular senescence may occur when some of the telomeres have shortened sufficiently to induce a DNA damage signal. Cancer cells escape the proliferative limits of replicative aging by up-regulating the expression of telomerase, an enzyme capable of adding telomere repeats to the ends of the chromosomes and maintaining their length. We have developed methods for identifying the presence of modified nucleotides in the subtelomeric DNA, for purifying telomeres (based on the presence of the 3' G-rich overhang) that yields a greater than 1,000-fold enrichment in a single step, for determining the size of the overhangs, and for measuring telomere sizes in interphase nuclei. These advances will permit us to address the following Specific Aims: 1) To understand the structure and function of base modifications in subtelomeric/telomeric DNA; 2) To determine what regulates the rate of telomere shortening; and 3) To define when and where cells with short telomeres accumulate in vivo in humans. Knowledge gained from these studies may lead to the ability to manipulate rates of telomere shortening, with consequences both for slowing cellular senescence and enhancing the efficacy of anti-telomerase cancer therapeutics. In addition, these investigations should identify the appropriate tissues and pathologies in which a causal relationship between replicative aging and organismal aging/disease can be tested experimentally, ultimately providing the basis for the development of direct therapeutic applications.